Indian Journal of Pharmacology Home 

[Download PDF]
Year : 2021  |  Volume : 53  |  Issue : 1  |  Page : 13--18

Determination of in vitro human whole blood-to-plasma ratio of THJ-018 utilizing gas chromatography–Mass spectrometry

Sachil Kumar1, Remal Nahar Khleel Alkhibery1, Abdulsallam Bakdash1, Mohamed Sultan Mohamed Abdelhady2,  
1 Department of Forensic Sciences, College of Criminal Justice, Naif Arab University for Security Sciences, Riyadh, Saudi Arabia
2 Riyadh Poisons Control & Medical Forensic Chemistry Center, Kaab Ibn Al Harith, Ar Rawdah, Riyadh, Saudi Arabia; Egyptian Drug Authority, National Organizations for Drug Control and Research (NODCAR), Giza, Egypt

Correspondence Address:
Dr. Sachil Kumar
Assistant Professor, Department of Forensic Sciences, College of Criminal Justice, Naif Arab University for Security Sciences, Riyadh
Saudi Arabia


OBJECTIVES: This study was aimed to determine in vitro human whole blood-to-plasma ratio (KWB/P) of THJ-018 by gas chromatography/mass spectrometry (GC/MS). MATERIALS AND METHODS: The samples (human blood) were sprayed with THJ-018 and an internal standard and extracted using solid-phase extraction. THJ-018 was determined in the final extracts by GC/MS. RESULTS: The value for KWB/P was 1.56 (1.38–1.81), and red blood cell partitioning was 1.01 (1.01–1.02). The distribution of THJ-018 between whole blood and plasma was observed to be affected by temperature. CONCLUSION: The data analysis supports the proposition that the ratio of the plasma to whole blood concentrations (1.56) is a suitable parameter characterizing THJ-018 distribution in whole blood. For toxicological analysis, it would be best to refrain from converting any drug concentration measured in whole blood to that anticipated in plasma or serum; however, toxic and therapeutic concentrations should be determined for the individual specimens collected.

How to cite this article:
Kumar S, Khleel Alkhibery RN, Bakdash A, Mohamed Abdelhady MS. Determination of in vitro human whole blood-to-plasma ratio of THJ-018 utilizing gas chromatography–Mass spectrometry.Indian J Pharmacol 2021;53:13-18

How to cite this URL:
Kumar S, Khleel Alkhibery RN, Bakdash A, Mohamed Abdelhady MS. Determination of in vitro human whole blood-to-plasma ratio of THJ-018 utilizing gas chromatography–Mass spectrometry. Indian J Pharmacol [serial online] 2021 [cited 2022 Nov 27 ];53:13-18
Available from:

Full Text


Endogenous cannabinoid receptors respond to synthetic cannabinoids (SCs), producing the same cannabimimetic effects as Δ9-tetrahydrocannabinol (THC).[1],[2] Toxicity may be caused by SC abuse, causing death, cardiac toxicity, stroke, rhabdomyolysis, and acute kidney injury.[3],[4] Despite these threats to health, SC consumption continues. To lure customers, vendors market these items in bright foil containers and plastic bags. Cloud 9, Spice Gold, K2/Spice, Spice Silver, Mojo, Mary Joy, Arizona, Joker, Bombay Blue, Black Mamba, Krypton, Kush, Lava Red, Blue Cheese, Banana Cream Nuke, Kronic, and Zohai are some of the brand names for these products.[14] Numerous SCs like JWH-073, AKB48, and XLR11 are classified as illegal drugs in nearly all European countries, the US, and Japan. SCs first were documented in Europe during the early 2000s, then in the United States in 2008.[12] However, compounds with more diverse structures continue to be produced and used.[24]

THJ-018 [1-naphthalenyl(1-pentyl-1H-indazol-3-yl)-methanone] and its 5-fluoro analog, THJ-2201 [(1-(5-fluoropentyl)-1H-indazol-3-yl)(naphthalen-1-yl)methanone] are two new SCs.[12] Some chemical suppliers sold it as a legal substitute for the well-known JWH-018, which was prohibited in 2009. In 2014, they were uncovered in Russia, Japan, and the US.[5],[6],[7],[8] Since January 2014, a total of 220 THJ-2201 reports have been documented by the National Forensic Laboratory Information System.[7] Despite the lack of official data on THJ-018 prevalence, both analogs appear to be popular, based on observation of drug-user forums.[9] In Japan, THJ-018 and THJ-2201 were listed as illegal substances in August 2014.[10] No pharmacological data for THJ-018 and THJ-2201 are available; however, based on their structures, it is expected that they will react with cannabinoid receptors in the same way as JWH-018 and 1-(5-fluoropentyl)-3-(1-naphthoyl) indole and matches much of the in vivo properties of Δ9-THC.[11] Drug-user forums highlight adverse effects such as anxiety, paranoia, psychosis, renal pain, and muscular spasm that are far more likely to occur at elevated doses and can cause death or injury.[9]

Like all SCs, chronic use of THJ-018 may cause moderate addiction.[22],[23] This substance has a high propensity for addiction, with some users developing extreme psychiatric or physical dependence as a result of its use. When users avoid using, they can experience cravings and withdrawal symptoms as a result of their addiction. Prolonged and repeated use of THJ-018 may result in a tolerance to its effects. No valid data exist on the toxicity of SCs; however, the naphthalene moiety found in THJ-018 and certain other SCs, which may induce toxicity or cancer, is a cause for concern.[18]

The type of biological matrix that is being sampled and analyzed may have an impact on the concentration of the analyte of interest. For instance, for some drugs, the concentration in plasma is different from the concentration in whole blood. Furthermore, the type of the analyte, variations in hematocrit, water content, and temperature can have an influence on the sample concentration, which leads to different whole blood-to-plasma ratios (KWB/P), for THC, as an example, this ratio is 0.6.[19],[25] Taking this into account, the partitioning behavior of compounds, the degree of binding, and the different distribution between the different types of samples should be considered in pharmacological and toxic cases.[20]

According to our knowledge, there is no research directly examining whole KWB/P for THJ-018. This study aims to determine the whole KWB/P of THJ-018 in samples of human blood and plasma and to examine the effect of incubation temperature on THJ-018 concentrations and the time course after incubation on this ratio. Furthermore, a precise and effective gas chromatography/mass spectrometry (GC/MS) system for determining THJ-018 in human blood was developed, optimized, and validated.

 Materials and Methods

Chemicals and reagents

In this experiment, all the reagents and chemicals employed were of high purity. THJ-018 and XLR-11 (internal standard) were purchased from Cerilliant (USA). Potassium dihydrogen phosphate, ethyl acetate, potassium hydroxide, and ammonia were obtained from Merck Millipore (Germany). Sigma-Aldrich supplied the dichloromethane and methanol.

Blood and plasma samples

Whole blood samples (2.5 mL) were collected from healthy individuals (single pool) in standard ethylenediaminetetraacetic acid-containing tubes (Becton Dickinson). Plasma was prepared by centrifugation for 10 min at a rate of 5000 rpm. After centrifugation, the plasma samples were placed in a clean polypropylene tube and held at −20°C until the analysis was carried out.

Sample preparation, incubation of drug in whole blood and control plasma

Hematocrit (H) was measured by a microhematocrit capillary assay. Control plasma (CP) was prepared by centrifugation from the same batch of whole blood. In 5 mL glass tubes, 3 mL prewarmed whole blood and CP were spiked with the selected concentrations of THJ-018 (25, 75, 150, 300, 500, and 750 ng/mL) and then incubated at the selected temperatures (−20, 4, 20 ± 2 [room temperature], and 37°C). At selected points of time (5, 30, 60, 120, and 180 min), the incubated whole blood was removed and plasma was prepared. Aliquots of the incubated CP were also removed at each time-point. One mL of plasma and 1 mL of control plasma were vortexed for 3 minutes after being fortified with 25 µL of a 2 ng mL-1 solution of XLR-11 (I.S.) and 2 mL of 100 mM pH 10.0 phosphate buffer. Post centrifugation (5 min at 5,000 rpm), the supernatant solution was placed into the solid-phase extraction (SPE) column. The red blood cell partitioning (KRBC/P) and KWB/P were calculated based on the following equations: KRBC/P = 1 + (1/H) ([CCP/CP]-1) and KWB/P = CCP/CP, where H is the hematocrit, CCP is the THJ-018 concentration in the CP, and CP is the THJ-018 concentration in the plasma separated from the incubated whole blood. The method has also been described by Wen et al.[21]

Solid-phase extraction

The SPE was conducted on CHROMABOND® C18 ec columns. The SPE cartridges were preconditioned as detailed by Bravo et al.[13] The prepared samples were then passed through the column at rates of between 1 and 1.5 mL/min. The sorbent was treated with 3 mL of 100 mM pH 10.0 phosphate buffer and 3 mL of water solution (50 mL water + 500 µL ammonia). The column was dried completely under a full vacuum at 15 mmHg for 5 min. Finally, the THJ-018 was eluted with 1.5 mL of dichloromethane/n-propanol/ammonia (78:20:2, v/v/v) into GC-Vials. The solvent was evaporated with a steady stream of nitrogen and residues were reconstructed in 50 µL ethyl acetate.

Gas chromatography–Mass spectrometry

The analysis was made using the Agilent 7000C-Triple-Quadrupole GC/MS system with 7890B Series gas chromatograph equipped with an Autosampler 7683 series. Separation of THJ-018 was conducted using a 5% phenyl-methylpolysiloxane phase (HP-5MS) column. Helium (purity 99%) was utilized as the carrier gas, with a constant flow of 1 mL/min. The injected volume was 1 μL and run in pulsed-split mode. The oven temperature was programmed in the following way: the initial temperature was set at 100°C, then raised to 240°C with a ramp of 12°C min–1, and then kept at 240°C for 18 min.

The temperature of the GC to MS transfer line was 290°C, and the electron impact (EI) ion source was 230°C. The mass spectra were operated in EI positive mode (70 eV). THJ-018 and I. S. detection was done using single-ion monitoring mode.

The subsequent ions were monitored:

for THJ-018 m/z 271 (quantifier), 127 and 324 (qualifier), for I.S. m/z 232 (quantifier), 144 and 247 (qualifier).

Determination was made on the basis of the ratio between the peak areas of THJ-018 to that of the I. S.

Statistical analysis

The one-way ANOVA is used to calculate the statistical difference between various groups. Tukey analysis test was done for comparison between the groups. Means and standard deviations for the relevant parameters have been determined. P values from all data analyses were presented and found to be statistically significant (generally ≤0.05).


Method validation

The system for analyzing THJ-018 in plasma utilizing SPE and GC-MS was developed and validated.[15] The retention times were 14.45 and 12.83 min for THJ-018 and I. S., respectively. To guarantee the high selectivity obtained, the run time was 18 min. The linearity of the method was determined in triplicate for each concentration in the range of 2.5–1000 ng/m, with a strong correlation coefficient (r2 = 0.9989). The method showed good sensitivity with the limit of detection (LOD) of 0.5 ng/mL and the limit of quantitation (LOQ) of 2.5 ng/mL based on the analysis of 1 mL plasma in ten replicates.

The extraction recovery was calculated by administering the full protocol to triplicate plasma samples in 3 days at two spiking levels between 50 and 500 ng/mL. The extraction recovery was expressed as recovery percentage. Proper extraction recovery (86.53% ± 7.24%) (n = 3) of THJ-018 from spiked plasma was reported, which was within the appropriate limit.

Accuracy and precision were studied by investigating the intra-assay and inter-assay. Inter-assay was calculated by measuring intraday data of three parallel injections of three independent concentrations of THJ-018 (10, 75, and 300 ng/mL). Intra-assay was based on interday data of three parallel injections of three independent concentrations of THJ-018 (10, 75, and 300 ng/mL). The recovery percentages were calculated. Accuracies (as % recovery) were 99.95% (95.84–101.24%) and 101.05% (96.72–112.31%) for the inter-assay and intra-assay, respectively. The precision values obtained for RSD% were 5.88% and 7.76% for the inter-assay and intra-assay, respectively.

Preliminary detection of blood-to-plasma ratio

The preliminary detection results of KWB/P and KRBC/P were investigated using 500 ng/mL concentration in blood and in CP samples (n = 12) which were incubated for 2 h at 37°C, mean hematocrit value was 43 vol%. [Table 1] shows the mean value KWB/P = 1.56 (1.38–1.81) with RSD% = 8.04 and the mean value KRBC/P = 1.01 (1.01 − 1.02) with RSD = 0.289.{Table 1}

As reported in [Table 1], the present experiments showed that THJ-018 exhibits an important accumulation in human whole blood rather than plasma fluid (KWB/P > 1.5) at 500 ng/mL concentration. According to these data, analysis of whole blood should be preferred with that of plasma for THJ-018 due to higher concentration with the purpose to increase the sensitivity and to obtain a lower limit of LOD and LOQ. Moreover, in the pharmacokinetic studies, sampling whole blood rather than plasma could be important to avoid overestimates of blood clearance.

Effect of incubation time on blood-to-plasma ratio

The effect of incubation time (5, 30, 60,120, and 180 min) using the concentration (300 ng/mL) at room temperature (20°C ± 2°C) on the THJ-018 KWB/P ratio in human blood (n = 6) is summarized in [Table 2]. The P value between groups was < 0.0001, considered significant. The highest mean KWB/P values were 1.68 ± 0.09 and 1.66 ± 0.08 at 120 min and 180 min, respectively, and lowest for 5 min, 1.36 ± 0.10.{Table 2}

The mean KWB/P values were highest in groups 180 and 120 min as compared to the other three groups, and it was statically significant between groups 5 min, 30 min, and the other groups, as shown in [Table 3] after one-way ANOVA test using Tukey analysis test. THJ-018 KWB/P ratio was not statically significant between groups 60, 120, and 180 min, which means the KWB/P values are stable after 1-h incubation, and the time course of the KWB/P ratio indicates that the parameter altered least after 1-h of incubation [Table 3].{Table 3}

Effect of concentration on blood-to-plasma ratio

The effect of concentration (25, 75, 150, 300, and 750 ng/mL) after incubation time (120 min) at room temperature (20°C ± 2°C) on blood partitioning are summarized in [Table 4]. Data have shown that there is no important impact of substance concentrations (25–750 ng/mL) on the KWB/P of THJ-018 [Table 4] and [Table 5].{Table 4}{Table 5}

Effect of temperature on blood-to-plasma ratio

Mean KWB/P values with temperature 37°C were 1.69 ± 0.06, for temperature 25°C, 1.63 ± 0.08, and lowest for temperature − 20°C, 1.52 ± 0.06. P < 0.004 is considered highly significant. Temperature course of the KWB/P ratio proves that the parameter changed [Table 6]. Tukey analysis test showed that incubation time −20°C versus 37°C showed significant values (P < 0.001).{Table 6}


A GC-MS system for identifying and quantifying THJ-018 in blood and plasma samples has been developed, validated, and then reliably used to calculate the KWB/P. Important parameters of method validation such as linearity, accuracy, precision, LOD, and LOQ were satisfactory.

In the present study, a straightforward technique affording in vitro measurement of drug distribution between whole blood and plasma was validated. This approach was based on measurement of time, temperature, and concentration dependence of THJ-018 distribution partition between whole blood and plasma.

Our experiment found that 2-h incubation at 37°C is sufficient to reach the equilibrium distribution of THJ-018 between whole blood and plasma. The temperature dependence showed an increase in THJ-018 accumulation in plasma with increasing temperature. On the other hand, no significant influence of various THJ-018 concentrations on whole blood/plasma ratio was observed. A nonsignificant increase in the whole KWB/P with high THJ-018 concentrations can be explained by gradual saturation of their binding to plasma proteins,[16] and it proves the previous described tricyclic antidepressant redistribution in the case of an acute overdose.[17]

Considering the measurement of distribution of THJ-018 in whole blood and in erythrocyte membrane suspension, we can conclude that the whole blood/plasma ratios to a large extent are characteristic of THJ-018. This can be explained first by the fact that THJ-018 in blood is, to a large extent, bound to proteins and to the lipid part of cell membranes and second by low plasma protein binding (PPB). From a pharmacokinetic perspective, as is known, drugs with high PPB are relatively protected from first-pass hepatic metabolism,[26] which means THJ-018 can undergo high metabolism with possible active metabolites[11] which explain its effect with low concentrations.

KWB/P is a crucial consideration for forecasting the pharmacokinetics of the entire body in conformity with other absorption, distribution, metabolism, excretion and toxicity (ADME-Tox) and physicochemical properties. Furthermore, forensic examinations are mostly conducted on whole blood, as there is often no serum available.[27]


The work described in this article for estimation of THJ-018 in human plasma reports a GC-MS method for analysis in human plasma. From the experimental data, it reflects that the distribution of THJ-018 between plasma and whole blood was observed to be affected by temperature. The data analysis supports the proposition that the ratio of the plasma to whole blood concentrations is a suitable parameter characterizing THJ-018 distribution in whole blood. The developed method is highly selective and successfully applied for the estimation of THJ-018 in human plasma to carry out a toxicological study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Castaneto MS, Gorelick DA, Desrosiers NA, Hartman RL, Pirard S, Huestis MA. Synthetic cannabinoids: Epidemiology, pharmacodynamics, and clinical implications. Drug Alcohol Depend 2014;144:12-41.
2Desrosiers NA, Lee D, Concheiro-Guisan M, Scheidweiler KB, Gorelick DA, Huestis MA. Urinary cannabinoid disposition in occasional and frequent smokers: Is THC-glucuronide in sequential urine samples a marker of recent use in frequent smokers? Clin Chem 2014;60:361-72.
3Hermanns-Clausen M, Kneisel S, Szabo B, Auwärter V. Acute toxicity due to the confirmed consumption of synthetic cannabinoids: Clinical and laboratory findings. Addiction 2013;108:534-44.
4Seely KA, Lapoint J, Moran JH, Fattore L. Spice drugs are more than harmless herbal blends: A review of the pharmacology and toxicology of synthetic cannabinoids. Prog Neuropsychopharmacol Biol Psychiatry 2012;39:234-43.
5Uchiyama N, Shimokawa Y, Kawamura M, Kikura-Hanajiri R, Hakamatsuka T. Chem analysis of a benzofuran derivative, 2-(2-ethylaminopropyl) benzofuran (2-EAPB), eight synthetic cannabinoids, five cathinone derivatives, and five other designer drugs newly detected in illegal products. Forensic Toxicol 2014;32:266-81.
6Shevyrin V, Melkozerov V, Nevero A, Eltsov O, Morzherin Y, Shafran Y. 3-Naphthoylindazoles and 2-naphthoylbenzoimidazoles as novel chemical groups of synthetic cannabinoids: Chemical structure elucidation, analytical characteristics and identification of the first representatives in smoke mixtures. Forensic Sci Int 2014;242:72-80.
7Drug Enforcement Administration, Department of Justice. Schedules of controlled substances: Temporary placement of three synthetic cannabinoids into schedule I. Final order. Fed Regist 2015;80:5042-7.
8Uchiyama N, Shimokawa Y, Matsuda S, Kawamura M, Kikura-Hanajiri R, Goda Y. Two new synthetic cannabinoids, AM-2201 benzimidazole analog (FUBIMINA) and (4-methylpiperazin-1-yl)(1-pentyl-1H-indol-3-yl) methanone (MEPIRAPIM), and three phenethylamine derivatives, 25H-NBOMe 3,4,5-trimethoxybenzyl analog, 25B-NBOMe, and 2C-N-NBOMe, identified in illegal products. Forensic Toxicol 2014;32:105-15.
9Drugs-Forum; 2014. Available from: [Last accesses on 2021 Mar 28].
10National Institute of Health Sciences. Data Search System for New Psychoactive Substances; 2015. Available from: [Last accessed on 2015 Mar 28].
11Chimalakonda KC, Seely KA, Bratton SM, Brents LK, Moran CL, Endres GW, et al. Cytochrome P450-mediated oxidative metabolism of abused synthetic cannabinoids found in K2/Spice: Identification of novel cannabinoid receptor ligands. Drug Metab Dispos 2012;40:2174-84.
12Guideline IH. Validation of analytical procedures: text and methodology. Q2 (R1). 2005;1(20):05.
13Amitai Y, Kennedy EJ, DeSandre P, Frischer H. Distribution of amitriptyline and nortriptyline in blood: Role of alpha-1-glycoprotein. Ther Drug Monit 1993;15:267-73.
14Amitai Y, Erickson T, Kennedy EJ, Leikin JB, Hryhorczuk DO, Noble J, et al. Tricyclic antidepressants in red cells and plasma: Correlation with impaired intraventricular conduction in acute overdose. Clin Pharmacol Ther 1993;54:219-27.
15Diao X, Wohlfarth A, Pang S, Scheidweiler KB, Huestis MA. High-resolution mass spectrometry for characterizing the metabolism of synthetic cannabinoid THJ-018 and its 5-fluoro analog THJ-2201 after incubation in human hepatocytes. Clin Chem 2016;62:157-69.
16Widman M, Agurell S, Ehrnebo M, Jones G. Binding of (+) and (-) D1-THC and (-)-7-hidroxy-d1THC to blood cells and plasma proteins in man. J Pharm Pharmacol 1974;26:914-6.
17Buxton IL. Pharmacokinetics and pharmacodynamics: The dynamics of drug absorption, distribution, action, and elimination. In: Goodman and Gilman's the Pharmacological Basis of Therapeutics. 11th ed.. New York: McGraw-Hill Medical Publishing Division; 2006.
18Wen Z, Huang Y, Behler N, Bambal R, Bhoopathy S, Owen A. Determination of red blood cell partitioning and whole blood to plasma ratio using human, rat, and mouse blood: Methods, model compounds and species differences. AAPSJ 2010;4305.
19Zimmermann US, Winkelmann PR, Pilhatsch M, Nees JA, Spanagel R, Schulz K. Withdrawal phenomena and dependence syndrome after the consumption of “spice gold”. Dtsch Arztebl Int 2009;106:464-7.
20Müller H, Sperling W, Köhrmann M, Huttner HB, Kornhuber J, Maler JM. The synthetic cannabinoid Spice as a trigger for an acute exacerbation of cannabis induced recurrent psychotic episodes. Schizophr Res 2010;118:309-10.
21Diao X, Huestis MA. New synthetic cannabinoids metabolism and strategies to best identify optimal marker metabolites. Front Chem 2019;7:109.
22Raes E, Verstraete A, Wennig R. Drugs and driving. In: Handbook of Analytical Separations. Vol. 6. BV: Elsevier Science; 2008. p. 611-51.
23Svennebring A. The impact of plasma protein binding on toxic plasma drug concentration. Int J Comput Biol Drug Design 2016;9:345-68.
24Jantos R. Comparison of the determination of drugs with influence on driving performance in serum, whole blood and dried blood spots. Toxichem Krimtech 2013;80:49-59.
25Bakdash A, AL-Mathloum AM, ElAmin EH, Taha NM, Kumar S, Nasr FA. Single-dose acute toxicity of THJ-2201 designer Cannabis drug: LD 50 and hematological and histological changes in mice. Egypt J Forensic Sci 2018;8:49.
26Bravo F, Gonzalez D, Benites J. Development and validation of a solid-phase extraction gas chromatography-mass spectrometry method for the simultaneous quantification of opioid drugs in human whole blood and plasma. J Chil Chem Soc 2011;56:799-802.
27Mills B, Yepes A, Nugent K. Synthetic cannabinoids. Am J Med Sci 2015;350:59-62.